Recently protease inhibitors were used to treat corneal ulceration and persistent epithelial defect. An organ culture system was used in this study to examine the effect of four protease inhibitors on rat corneal epithelial migration and on incorporation of 3H-leucine into trichloroacetic acid-precipitable material as an indicator of protein synthesis. The effect of the protein synthesis inhibitor, verrucarin A, on both parameters also was examined. These results showed that epithelial migration was dependent linearly on protein synthesis with inhibition ranging from 100% at 10(-5) M to 0% at 10(-8) M. Pepstatin A, an acid protease inhibitor, had no effect on migration at concentrations of 10(-5) M and 10(-6) M. At 0.5 mM, 1,10-phenanthroline, a metalloprotease inhibitor, blocked epithelial migration at 100% in a dose-dependent manner with an equivalent reduction in 3H-leucine incorporation. Aprotinin blocked migration 74% and 71% at 500 U/ml and 50 U/ml, respectively, whereas protein synthesis decreased only 41% and 21%, respectively. At 1.0 mM and 0.5 mM, phenylmethylsulfonyl fluoride (PMSF) blocked migration 92% and 71% and lowered protein synthesis 74% and 55%, both respectively. These data suggest that the serine proteases may be involved directly in epithelial migration because their inhibitors, aprotinin and PMSF, blocked it to such an extent that this finding cannot be explained by inhibition of protein synthesis alone.